A new view of responses to first-time barefoot running.

We examined acute alterations in gait and oxygen cost from shod-to-barefoot running in habitually-shod well-trained runners with no prior experience of running barefoot. Thirteen runners completed six-minute treadmill runs shod and barefoot on separate days at a mean speed of 12.5 km·h-1. Steady-state oxygen cost in the final minute was recorded. Kinematic data were captured from 30-consecutive strides. Mean differences between conditions were estimated with 90% confidence intervals. When barefoot, stride length and ground-contact time decreased while stride rate increased. Leg-and vertical stiffness and ankle-mid-stance dorsi-flexion angle increased when barefoot while horizontal distance between point of contact and the hip decreased. Mean oxygen cost decreased in barefoot compared to shod running (90% CI -11% to -3%) and was related to change in ankle angle and point-of-contact distance, though individual variability was high (-19% to +8%). The results suggest that removal of shoes produces an alteration in running gait and a potentially-practically-beneficial reduction in mean oxygen cost of running in trained-habitually-shod runners new to running barefoot. However, high variability suggests an element of skill in adapting to the novel task and that caution be exercised in assuming the mean response applies to all runners

Multi-omic profiling of EPO-producing Chinese hamster ovary cell panel reveals metabolic adaptation to heterologous protein production

Chinese hamster ovary (CHO) cells are the preferred production host for many therapeutic proteins. The production of heterologous proteins in CHO cells imposes a burden on the host cell metabolism and impact cellular physiology on a global scale. In this work, a multi‐omics approach was applied to study the production of erythropoietin (EPO) in a panel of CHO‐K1 cells under growth‐limited and unlimited conditions in batch and chemostat cultures. Physiological characterization of the EPO‐producing cells included global transcriptome analysis, targeted metabolome analysis, including intracellular pools of glycolytic intermediates, NAD(P)H/NAD(P)(+), adenine nucleotide phosphates (ANP), and extracellular concentrations of sugars, organic acids, and amino acids. Potential impact of EPO expression on the protein secretory pathway was assessed at multiple stages using quantitative PCR (qPCR), reverse transcription PCR (qRT‐PCR), Western blots (WB), and global gene expression analysis to assess EPO gene copy numbers, EPO gene expression, intracellular EPO retention, and differentially expressed genes functionally related to secretory protein processing, respectively. We found no evidence supporting the existence of production bottlenecks in energy metabolism (i.e., glycolytic metabolites, NAD(P)H/NAD(P)(+) and ANPs) in batch culture or in the secretory protein production pathway (i.e., gene dosage, transcription and post‐translational processing of EPO) in chemostat culture at specific productivities up to 5 pg/cell/day. Time‐course analysis of high‐ and low‐producing clones in chemostat culture revealed rapid adaptation of transcription levels of amino acid catabolic genes in favor of EPO production within nine generations. Interestingly, the adaptation was followed by an increase in specific EPO productivity. Biotechnol. Bioeng. 2015;112: 2373–2387. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc

Physiology of Saccharomyces cerevisiae Producing Recombinant Insulin in Continuous Culture

The yeast Saccharomyces cerevisiae has widely been used as a host for the production of heterologous proteins. High-level production of heterologous proteins is likely to impose a metabolic burden on the host cell and can thus affect various aspects of cellular physiology. Different target proteins often do not result in similar secretion yields, underlining the dependency of secretion efficiency on the physicochemical properties of the protein of interest. Two human insulin analogue precursors (IAPs) were used as model secretory proteins. The IAPs had minor differences in their amino acid sequences, yet pictured more than 7-fold difference in their secretion yields. Global transcriptome analysis carried out in aerobic glucose-limited chemostat experiments pinpointed distinct steps during the protein maturation pathway to be differentially regulated, and indicated an increased degradation of the IAP with the low secretion yield. The use of auxotrophic strains for improved IAP production was examined. The incremental truncation of the promoter of the auxotrophic marker gene URA3 led to higher plasmid copy numbers, and illustrated that the modification of the level of the recombinant gene dosage via the degree of promoter truncation can be a strong tool for optimizing the IAP productivity. The dynamic character of adaptive responses of S. cerevisiae towards a change in their nutrient access was studied based on global gene expression analysis, scrutinizing the impact of restricted supply of phosphate on the physiological state of IAP-expressing cells. The gradual decrease of the phosphate supply resulted in a step-wise modulated phenotypic response, thereby alternating the specific productivity and the secretory flux. A data-driven approach was applied to study the secretory IAP production in S. cerevisiae in prolonged chemostat cultures (80 generations), with the aim to explore the metabolic adaptation of the cells towards the burden of IAP production. Time-course analysis of global transcriptome and targeted metabolome analysis indicated at metabolic re-modeling of the recombinant cells based on augmented negative selection pressure on glycolytic overcapacity, changes in amino acid and central carbon metabolism, and mitochondrial dysfunction to account for decreased cellular expression efficiency in long-term chemostat cultures

Physiology of Saccharomyces cerevisiae Producing Recombinant Insulin in Continuous Culture

The yeast Saccharomyces cerevisiae has widely been used as a host for the production of heterologous proteins. High-level production of heterologous proteins is likely to impose a metabolic burden on the host cell and can thus affect various aspects of cellular physiology. Different target proteins often do not result in similar secretion yields, underlining the dependency of secretion efficiency on the physicochemical properties of the protein of interest. Two human insulin analogue precursors (IAPs) were used as model secretory proteins. The IAPs had minor differences in their amino acid sequences, yet pictured more than 7-fold difference in their secretion yields. Global transcriptome analysis carried out in aerobic glucose-limited chemostat experiments pinpointed distinct steps during the protein maturation pathway to be differentially regulated, and indicated an increased degradation of the IAP with the low secretion yield. The use of auxotrophic strains for improved IAP production was examined. The incremental truncation of the promoter of the auxotrophic marker gene URA3 led to higher plasmid copy numbers, and illustrated that the modification of the level of the recombinant gene dosage via the degree of promoter truncation can be a strong tool for optimizing the IAP productivity. The dynamic character of adaptive responses of S. cerevisiae towards a change in their nutrient access was studied based on global gene expression analysis, scrutinizing the impact of restricted supply of phosphate on the physiological state of IAP-expressing cells. The gradual decrease of the phosphate supply resulted in a step-wise modulated phenotypic response, thereby alternating the specific productivity and the secretory flux. A data-driven approach was applied to study the secretory IAP production in S. cerevisiae in prolonged chemostat cultures (80 generations), with the aim to explore the metabolic adaptation of the cells towards the burden of IAP production. Time-course analysis of global transcriptome and targeted metabolome analysis indicated at metabolic re-modeling of the recombinant cells based on augmented negative selection pressure on glycolytic overcapacity, changes in amino acid and central carbon metabolism, and mitochondrial dysfunction to account for decreased cellular expression efficiency in long-term chemostat cultures

The impact of phosphate scarcity on pharmaceutical protein production in <it>S. cerevisiae</it>: linking transcriptomic insights to phenotypic responses

Abstract Background The adaptation of unicellular organisms like Saccharomyces cerevisiae to alternating nutrient availability is of great fundamental and applied interest, as understanding how eukaryotic cells respond to variations in their nutrient supply has implications spanning from physiological insights to biotechnological applications. Results The impact of a step-wise restricted supply of phosphate on the physiological state of S. cerevisiae cells producing human Insulin was studied. The focus was to determine the changes within the global gene expression of cells being cultured to an industrially relevant high cell density of 33 g/l cell dry weight and under six distinct phosphate concentrations, ranging from 33 mM (unlimited) to 2.6 mM (limited). An increased flux through the secretory pathway, being induced by the PHO circuit during low Pi supplementation, proved to enhance the secretory production of the heterologous protein. The re-distribution of the carbon flux from biomass formation towards increased glycerol production under low phosphate led to increased transcript levels of the insulin gene, which was under the regulation of the TPI1 promoter. Conclusions Our study underlines the dynamic character of adaptive responses of cells towards a change in their nutrient access. The gradual decrease of the phosphate supply resulted in a step-wise modulated phenotypic response, thereby alternating the specific productivity and the secretory flux. Our work emphasizes the importance of reduced phosphate supply for improved secretory production of heterologous proteins.</p

The impact of phosphate scarcity on pharmaceutical protein production in S. cerevisiae: linking transcriptomic insights to phenotypic responses

Background:The adaptation of unicellular organisms like Saccharomyces cerevisiae to alternating nutrient availability is of great fundamental and applied interest, as understanding how eukaryotic cells respond to variations in their nutrient supply has implications spanning from physiological insights to biotechnological applications.Results:The impact of a step-wise restricted supply of phosphate on the physiological state of S. cerevisiae cells producing human Insulin was studied. The focus was to determine the changes within the global gene expression of cells being cultured to an industrially relevant high cell density of 33 g/l cell dry weight and under six distinct phosphate concentrations, ranging from 33 mM (unlimited) to 2.6 mM (limited). An increased flux through the secretory pathway, being induced by the PHO circuit during low Pi supplementation, proved to enhance the secretory production of the heterologous protein. The re-distribution of the carbon flux from biomass formation towards increased glycerol production under low phosphate led to increased transcript levels of the insulin gene, which was under the regulation of the TPI1 promoter.Conclusions:Our study underlines the dynamic character of adaptive responses of cells towards a change in their nutrient access. The gradual decrease of the phosphate supply resulted in a step-wise modulated phenotypic response, thereby alternating the specific productivity and the secretory flux. Our work emphasizes the importance of reduced phosphate supply for improved secretory production of heterologous proteins

Modulating heterologous protein production in yeast: the applicability of truncated auxotrophic markers

The use of auxotrophic Saccharomyces cerevisiae strains for improved production of a heterologous protein was examined. Two different marker genes were investigated, encoding key enzymes in the metabolic pathways for amino acid (LEU2) and pyrimidine (URA3) biosynthesis, respectively. Expression plasmids, carrying the partly defective selection markers LEU2d and URA3d, were constructed. Two CEN.PK-derived strains were chosen and insulin analogue precursor was selected as a model protein. Different truncations of the LEU2 and URA3 promoters were used as the mean to titrate the plasmid copy number and thus the recombinant gene dosage in order to improve insulin productivity. Experiments were initially carried out in batch mode to examine the stability of yeast transformants and to select high yielding mutants. Next, chemostat cultivations were run at high cell density to address industrial applicability and long-term expression stability of the transformants. We found that the choice of auxotrophic marker is crucial for developing a yeast expression system with stable heterologous protein production. The incremental truncation of the URA3 promoter led to higher plasmid copy numbers and IAP yields, whereas the truncation of the LEU2 promoter caused low plasmid stability. We show that the modification of the level of the recombinant gene dosage by varying the degree of promoter truncation can be a strong tool for optimization of productivity. The application of the URA3d-based expression systems showed a high potential for industrial protein production and for further academic studies

Modulating heterologous protein production in yeast: the applicability of truncated auxotrophic markers

The use of auxotrophic Saccharomyces cerevisiae strains for improved production of a heterologous protein was examined. Two different marker genes were investigated, encoding key enzymes in the metabolic pathways for amino acid (LEU2) and pyrimidine (URA3) biosynthesis, respectively. Expression plasmids, carrying the partly defective selection markers LEU2d and URA3d, were constructed. Two CEN.PK-derived strains were chosen and insulin analogue precursor was selected as a model protein. Different truncations of the LEU2 and URA3 promoters were used as the mean to titrate the plasmid copy number and thus the recombinant gene dosage in order to improve insulin productivity. Experiments were initially carried out in batch mode to examine the stability of yeast transformants and to select high yielding mutants. Next, chemostat cultivations were run at high cell density to address industrial applicability and long-term expression stability of the transformants. We found that the choice of auxotrophic marker is crucial for developing a yeast expression system with stable heterologous protein production. The incremental truncation of the URA3 promoter led to higher plasmid copy numbers and IAP yields, whereas the truncation of the LEU2 promoter caused low plasmid stability. We show that the modification of the level of the recombinant gene dosage by varying the degree of promoter truncation can be a strong tool for optimization of productivity. The application of the URA3d-based expression systems showed a high potential for industrial protein production and for further academic studies